Qualification & quantification de microparticles

Analytical Platform development for Microparticles detection, qualification and quantification

Biological context: Microparticles (MPs), present in various biological fluids such as blood, are small phospholipid microvesicles, arising from cell membrane via a process called vesiculation or budding, which present numerous and different concentrations, sizes, shapes, patterns of cell-surface and intracellular proteins as well as internal molecular compositions. MPs may originate from most of the cells, and are now recognized as specific structures that have multiple roles in the human body, and that are of pathophysiological relevance for several clinical disorders. Based on these statements, qualification and quantification of these MPs are crucial as they may serve as potential biomarkers of diseases or to qualify blood products.

Functional effects of MPs / MPs and pathologies:Moreover, if these better characterized MP subsets could be recovered selectively, a MP specific functional study would be possible on target cells of interest. In physiological conditions, platelet-derived MPs (PMP) are by far the most abundant in the bloodstream, representing about 70-90% of all circulating MPs. Increased levels of MPs present in plasma have been reported in several pathological situations (including thrombotic, cardiovascular or autoimmune diseases) to be associated with pro-thrombotic or pro-inflammatory activities. Accumulation of PMP in labile blood products (i.e., frozen fresh plasma and platelet concentrates) due to different steps of their process has been reported. Whether these PMP are involved in the side effects associated with transfusion remains to be determined. MPs produced from platelets in steady state exert rather anti-inflammatory properties while PMP produced in response to thrombin are pro-inflammatory and highly pro-thrombotic.

Analytical solutions, limitations and current challenges: Today, flow cytometry is the standard technique used to quantify circulating MPs and to determine their size. Nevertheless, flow cytometry is unable to provide accurate measurements of objects below 300 nm of diameter, due to the wavelength limitation. The monitoring of such multi-parameters, from MP size to their quantification and protein composition, at the nanometer resolution through a “label-free” analytical platform and in a potential multiplex format, is a real challenge for the whole scientific and medical community.

Contribution of our group to this field: The “label-free” analytical platform we developed consists in combining mainly Surface Plasmon Resonance (SPR) analysis, Atomic Force Microscopy (AFM) and mass spectrometry (MS) approaches. SPR can detect and quantify, in real-time, on a surface, molecular interactions, even in complex media. Such SPR biosensors are also expedient to the capture of natural soluble objects of interest by a chip surface presenting region of interest or arrays [Remy-Martin F. et al, (2012) Anal. Bioanal. Chem. 404(2), 423-432 ; Rouleau A. et al (2012), Sensors, 12(11), 15119-1513]. We also propose to lay on our expertise in atomic force microscopy (AFM) [Ewald M. et al (2015), Nanotechnology, 25(29) 295101 ; Heu C. et al (2012), J. Struct Biol, 178(1), 1-7 ; Berthier A. et al (2011) J. Molec. Recogn. 24(3), 429-435], to propose a deep MPs characterization in size and shape. Moreover, MS engaged on the chip after the MPs selective capture on arrays is the way to reach the sample protein signatures, in the different physio-pathological MPs conditions. The coupling of SPR with AFM and MS is a promising way to perform a correlation between an average measurement at the macroscale of MP biological activity on the chip and the in situ visualization of nanoscale biological events and protein signatures. In parallel, we developed size calibration standards, that cover the size range of MPs, in order to confirm that the chip reveals the whole content of the liquid sample we have to qualify [Obeid S. et al, (2017) Biosens. Bioelectron. 2017, 93:250-259].